Our major hypothesis is that regulation of sleep and wakefulness is coupled to regulation to cerebral energy stores and production of ATP. At the onset of wakefulness, we propose mechanisms are activated to increase energy supply and production and meet the increased energy demands. With continued wakefulness, changes at the transcriptional level decrease energy production and reduce ATP levels. As a result, adenosine accumulates and promotes sleep. This intuitively appealing idea has until recently been lacking experimental evidence, but preliminary data that we present supports this concept. In Specific Aim 1, we will test our hypothesis in both mouse and Drosophila with regard to the regulation of glycogen storage. We believe that early in wakefulness, the regulation of the activity of both the degradative and synthetic enzyme for glycogen (glycogen phosphorylase GP and glycogen synthase GS) are regulated in a coordinated way by phosphorylation to increase glycogen degradation and enhance energy supply. With more prolonged wakefulness, regulation at the transcriptional level alters the mRNA for both genes (GP, GS) as well as for protein targeting to glycogen, such that less glucose is made available from glycogen. In Specific Aim 2 we will broaden our evaluation of other sources of energy and of ATP-producing molecules. This aim is based on a hypothesis derived from microarray studies conducted by us. We propose that there is coordinated up-regulation of genes involved in the production of ATP, but this upregulation is only transient, returning towards baseline after a few hours. We will combine the high-throughput technology of real-time RT-PCR to evaluate, in multiple brain regions, specific candidate genes in relationship to sleep/wakefulness as suggested by our microarray studies. We will also quantify the protein products of selected genes and will study enzyme activity for a key mitochondrial enzyme that we have found is altered during wakefulness. These studies thus include comprehensive gene profiling; extensive temporal profiles; spatial localization within the brain; and studies of molecular regulation from the transcriptional to the post-translational level. These studies will be conducted in mice (Specific Aim 2). Finally, we will test the hypothesis that the transcriptional regulation of energy genes is mediated, at least in part, by the transcription factor cyclic-AMP response element binding protein (CREB). In Specific Aim 3, we will determine whether the "sleepy" Creb-deficient mice have deficits in expression of relevant energy genes that temporally correlate with their recently demonstrated inability to stay awake. Together with the results from Project 04, we will begin to establish whether--and how--the dynamic regulation of these genes could be part of the signaling mechanism for sleepiness and sleep promotion.